Photoacoustic imaging for cutaneous melanoma assessment: a comprehensive review

Abstract. Significance Cutaneous melanoma (CM) has a high morbidity and mortality rate, but it can be cured if the primary lesion is detected and treated at an early stage. Imaging techniques such as photoacoustic (PA) imaging (PAI) have been studied and implemented to aid in the detection and diagnosis of CM. Aim Provide an overview of different PAI systems and applications for the study of CM, including the determination of tumor depth/thickness, cancer-related angiogenesis, metastases to lymph nodes, circulating tumor cells (CTCs), virtual histology, and studies using exogenous contrast agents. Approach A systematic review and classification of different PAI configurations was conducted based on their specific applications for melanoma detection. This review encompasses animal and preclinical studies, offering insights into the future potential of PAI in melanoma diagnosis in the clinic. Results PAI holds great clinical potential as a noninvasive technique for melanoma detection and disease management. PA microscopy has predominantly been used to image and study angiogenesis surrounding tumors and provide information on tumor characteristics. Additionally, PA tomography, with its increased penetration depth, has demonstrated its ability to assess melanoma thickness. Both modalities have shown promise in detecting metastases to lymph nodes and CTCs, and an all-optical implementation has been developed to perform virtual histology analyses. Animal and human studies have successfully shown the capability of PAI to detect, visualize, classify, and stage CM. Conclusions PAI is a promising technique for assessing the status of the skin without a surgical procedure. The capability of the modality to image microvasculature, visualize tumor boundaries, detect metastases in lymph nodes, perform fast and label-free histology, and identify CTCs could aid in the early diagnosis and classification of CM, including determination of metastatic status. In addition, it could be useful for monitoring treatment efficacy noninvasively.


Introduction
2][3][4][5] Melanoma is the fifth most common cancer in the United States, with high morbidity and mortality. 6][9][10] Melanoma tumor depth is an important prognostic factor, along with ulceration status. 11Early detection and diagnosis is critical. 12The 5-year survival rate is 99% for localized melanomas but decreases to 63% with regional metastases and 20% with distant metastases. 8iopsies have long been considered the diagnostic standard for melanoma. 13,14Performing biopsies can result in pain, anxiety, scarring, and disfigurement for patients, as well as a considerable cost to the healthcare system.Given the high mortality associated with melanoma depth and metastasis, there is an urgent need for accurate, noninvasive methods to detect and monitor the disease.2][43] Beyond their capabilities for an initial diagnosis, other opportunities for melanoma staging through imaging involve analysis of lymph nodes for metastases and detection of circulating (melanoma) tumor cells (CTCs).
4][55][56][57][58] This induces the generation of PA signals, which are then detected by an ultrasound (US) transducer and reconstructed into a two-dimensional (2D) or three-dimensional (3D) image.The strength of the PA signals correlates with tissue chromophore absorption properties, which depend on the wavelength and the chromophore's absorption spectrum.The combination of using light illumination and US detection provides PAI with advantages (e.g., high sensitivity and specificity at a greater imaging depth) over other modalities that utilize only light or US for signal generation and detection. 39,594][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79] US imaging provides improved penetration depth 80 but has limited ability to differentiate between skin conditions. 81AI can be classified by axial resolution of generated images into PA macroscopy, mesoscopy, and microscopy.3][84] This has enabled noninvasive imaging of breast cancer, 85 Crohn's disease activity, 86 brown fat metabolism, 87 and various blood vessels located in the neck (carotid), 88 hands, 89 and feet, 90 as well as whole-body imaging in animals. 91,92icroscopy utilizes higher US frequencies and allows for imaging with resolution capable of imaging individual cells [93][94][95][96] but with much lower penetration depth.Mesoscopy is a bridge between macroscopy and microscopy, utilizing US frequencies that image at penetration depths of several millimeters, 82 making it optimal for dermatological investigations. 97Mesoscopy has been used to visualize: skin morphology generally, 87,98,99 vascular patterns, morphology in psoriasis and atopic dermatitis, 100 nailbed microvasculature, 101 and vasodilation induced by hyperthermia. 102nother way to classify PAI systems is based on how the image is reconstructed (Fig. 1): in PA tomography (PAT), also known as PA computed tomography (PACT), a reconstruction algorithm is used to convert the PA signals collected at different locations/angles around the object into an image, 47,48,103,104 whereas in raster scan PAI, the pixels/voxels of the image are generated as the sample is raster scanned.Based on the arrangement of transducers, PAT can further be categorized into single element PAT, linear array PAT (LA-PAT), ring array PAT, and hemispherical PAT, 47,48,103,105,106 but LA-PAT is more widely implemented than other forms.Raster scan PAI has also been implemented in a variety of configurations, 47,[107][108][109] which can be divided into optical resolution PA microscopy (OR-PAM) and acoustic resolution PA microscopy (AR-PAM).1][112] With OR-PAM, reaching to the cellular level resolution is achievable; however, the imaging depth is limited to a few millimeters. 108In AR-PAM, a spherically focused transducer scans the sample. 110,113,114This yields a greater penetration depth than OR-PAM but with a coarser resolution.Raster scanning optoacoustic mesoscopy (RSOM) is an implementation of AR-PAM that has also been used for skin imaging.Because raster scanning images samples point by point, image acquisition can be slow.Configurations for different PA systems (PACT, RSOM, OR-PAM, and AR-PAM) are shown in Fig. 1.
A third way to categorize PAI systems is whether they use a single wavelength laser to collect images or multiple wavelengths.In general, a single wavelength cannot distinguish between different chromophores. 92In multispectral PAI (MPAI), two or more wavelengths 92,[115][116][117][118][119] are used to differentiate between chromophores.For instance, melanin and hemoglobin can be differentiated one from another using 584 and 764 nm. 115,116,120MPAI has been used to study numerous diseases, including prostate cancer, 121 glioblastoma, 122,123 ovarian cancer, 124 thyroid disease, 125,126 Crohn's disease, 86 liver disease, 127 cardiac injury, 128 systemic sclerosis, 129 and cutaneous tumors. 130,131n terms of applications, PAI can also be categorized with regards to the type of questions that the imaging modality can be used to answer, namely, fitness of each PAI system to determine: (a) melanoma detection and depth measurement, (b) tumor angiogenesis, (c) lymph node metastases, (d) CTCs, and (e) virtual histology (see Fig. 2).Exogenous contrast agents have also been applied for some of these applications.Hardware designs of selected PAI systems that have been used in some of these applications are shown in Fig. 3.This categorization is consistent with the organization of the paper.

Melanoma Detection and Depth Measurement
Imaging techniques able to detect melanoma with high sensitivity and specificity could greatly reduce the number of biopsies that are currently performed.Once a melanoma is confirmed through detection, the next step is melanoma staging.Staging melanoma is critical since it determines prognosis and treatment options. 12Melanoma depth (Breslow depth) is one of the two variables used to stage localized melanoma; 12 the other one is ulceration status.The tumor (T) category of the tumor-node-metastasis staging system depends on tumor depth and is classified as T1 (≤1.00 mm), T2 (1.01 to 2.00 mm), T3 (2.01 to 4.00 mm), or T4 (>4.00 mm). 12or T1 melanomas, the 10-year survival rate is ∼92%, but drops to 50% for T4 melanomas. 12][248][249][250] Imaging modalities could be used to stage melanoma, guide surgical planning, and prevent incomplete excisions and subsequent additional surgeries.

Animal studies
Oh et al. 116 used dual-wavelength AR-PAM to detect melanoma in mice inoculated with highly invasive B16 skin melanoma cells.Due to the difference in peak optical absorption between melanin and hemoglobin, a near-infrared (NIR) light (764 nm) and a visible light (584 nm) were used to visualize the melanoma [see Fig. 4(a)(i)] and surrounding vascular structures [see Fig. 4(a)(ii)].Figures 4(a)(iii) and 4(a)(iv) illustrate the B-scan images across the red dotted line in Figs.4(a)(i) and 4(a)(ii).The maximum thickness of the melanoma was found to be 0.5 mm.Zhou et al. 187,188 conducted several experiments to study PAI in nude mice using a similar melanoma model.They used a handheld AR-PAM to measure the depth of melanoma. 188g. 2 PAI applications for characterizing melanoma, discussed in this review.CTC, circulating tumor cells; PAI, photoacoustic imaging.Their PAM utilized an "annular-shaped" light illumination method, in which light (with 8 mm inner diameter and 20 mm outer diameter) bypasses the center of the tumor and instead is delivered in the direction normal to the surface. 188The melanoma depth (3.66 mm) as measured by their system [see Fig. 4(b)] corresponded well to the actual, postexcisional thickness (3.75 mm). 188In another experiment utilizing a LA-PAT system, tumor depth and volume were measured and revealed an increase in tumor depth and volume from day 3 [Fig.4(c)(i)] to day 6 [Fig.4(c)(ii)] after injection with the B16 cells. 187The depth increased from 1.32 to 2.77 mm and volume increased from 22.365 to 71.931 mm 3 .Moreover, the system was able to measure the rate of growth for both depth and volume of tumor.Recently, Wang et al. 184

Human studies
In a pilot study, Zhou et al. 135 analyzed 10 melanomas in 7 patients using a LA-PAT system [see Fig. 5(a)] immediately preceding excisional biopsy.Melanomas at depths from 0.2 to 6 mm were visualized, but deeper tumors were beyond the detection limit of the LA-PAT system.Figure 5(a) shows the resultant PA images [see Figs.(a)(iii) and 5(a)(vi)] of two patients, one with CM metastases on the left lower extremity and the other with primary acral lentiginous melanoma on the right foot.Histological images acquired from both patients showed that the detected melanoma depth was consistent with actual Breslow depth, showing the promising capability of the system for detection.Kim et al. 177 developed an integrated PA and US imaging (PAUSI) system by combining a clinical US machine and a multispectral portable laser.The imaging system was utilized to image a patient's melanoma ex vivo after excision [see Fig.  the 680-nm laser.Moreover, PA imaging indicated areas of melanoma that are not visible to the surgeon [yellow triangles in Fig. 5(b)(ii)].In addition, the measured thickness of the melanoma region (420 AE 320 μm) matched well with the histopathological results.A larger study on PAI was conducted by Breathnach et al. 115 using LA-PAT and spectral unmixing, to pre-operatively image 32 pigmented lesions suspicious for melanoma in 27 patients.With spectral unmixing, they separated the absorption signature of melanin-containing cells and mapped the spatial distribution of it based on this signature, because the absorption spectrum of melanin varies within the NIR region.The lesion depths measured by PA were highly correlated with histopathologic measurements, with a correlation coefficient of 0.98 for benign lesions and 0.99 for melanomas.Using the same PA probe, the lesion architecture, adnexal depth (depth of lesion extension into the skin appendages), and various skin layers were also imaged, allowing for differentiation of superficial from invasive lesions based on their dermal-epidermal junction penetration, i.e., lesion penetrated through the dermal-epidermal junction [see Fig. 5(c)].According to the authors, due to the tissue sample dehydration and loss of skin tension in vivo, PAI overestimated lesion depth as compared with histopathology.Recently, Park et al. 133 utilized a 3D multispectral PAT system to noninvasively measure depth and outline the boundary of melanomas for optimal surgical margin selection.Six melanoma patients were examined.They imaged melanoma of various forms, sizes (1.3 to 30 mm for lateral diameter and 0.6 to 9.1 mm for depth), and locations (sole, chest, thigh, heel, and palm) using their multispectral PA/US system [see Fig. 5(d)].For five of the six case studies, melanoma depth was measured using multispectral analysis and confirmed a high correlation against histopathologic results with a mean absolute error of 0.36 mm.In a signal-based study, Swearingen et al. 252 investigated if label-free MPAI could distinguish vascular from pigmented (melanotic) lesions in 15 human The experiment proved the ability of MPAI to distinguish between vascular and pigmented lesions.About 15 lesions were biopsied after imaging, revealing 8 vascular and 7 pigmented lesions.Data analysis was carried out via two statistical methods, the classical method (standard multivariate analysis classification techniques) and a Bayesianmodel-based approach.The classical method attained a perfect lesion diagnosis rate, whereas the Bayesian approach had a 20% error rate.
Figures 4 and 5 and the related text describe results from a selection of PAI studies of melanoma detection and depth measurement.Table 1 includes specific characteristics of the PAI systems used in published studies on melanoma detection/depth measurement for both animal and human studies.

Measurement of Tumor Angiogenesis in Melanoma
Tumor angiogenesis refers to the formation of new blood vessels within a tumor, or the growth of blood vessels between a tumor and its surrounding tissues.Tumor-associated vasculature not only regulates the supply of nutrients and oxygen to the tumor but also expedites tumor invasion and metastasis.Therefore, as an essential indicator of disease progression, vascularization within a tumor can be used to assess the potential for metastasis. 184,253PAI has shown great promise in monitoring the progression of tumor angiogenesis 143,254 using OR-PAM and also RSOM in mice and humans (Table 2 and Fig. 6).

Animal studies
In one study, Omar et al. 182 utilized RSOM to visualize angiogenesis and tumor growth in melanomas of mice in vivo over several days.B16F10 melanoma cells were injected subcutaneously into the mammary fat pad of Hsd: Athymic Nude-Foxn1 nu mice, and tumor growth and angiogenesis were monitored using spherically focused US detectors with central frequencies of 50 and 100 MHz.The 50 MHz detector was superior in imaging larger structures such as larger, oblique blood vessels [see Fig.Each subfigure in Fig. 6(b) includes an inset taken from the proximity of the tumor.The growth of the tumor from day 2 to day 9 was illustrated by the growth of the black nonvascularized spot, indicated by a thick arrow in all the subfigures in Fig. 6(b).Moreover, in each inset, it is observed that upon interaction with the tumor, the two big vessels (denoted by a thinner arrow) start rearranging; at the same time, smaller vessels start growing in that region, clearly representing tumor angiogenesis at a microvasculature level.Zhao et al. 185 utilized OR-PAM at two wavelengths (570 and 1064 nm) to study melanoma tumor angiogenesis.B16 melanoma cells were injected subcutaneously into the mouse ear and tumors were imaged on day 9 [see Fig. 6(c)(i)], day 13 [see Fig. 6(c)(ii)], day 14 [see Fig. 6(c)(iii)], and day 15 [see Fig. 6(c)(iv)] after tumor inoculation in two mice.In the earlier day postinoculation, small diameter (<25 to 30 μm) vessels were most prevalent.On the following 2 days, the number of large diameter vessels (50 to 95 μm) increased while the proportion of small diameter vessels decreased.On day 15, vessels with diameter >100 μm were visualized.Moreover, the vessel density, vessel tortuosity, and fractional dimension (quantitative parameters they used to assess tumor growth) also showed an overall upward trend from day 9 to day 15.Thus, the authors concluded that with the growth of melanoma, the vascular networks become stronger and complex, which was consistent with visual results in Fig. 6(c).Another study by Zhou et al. 186 used a combined all optical PA microscopy system and RCM system to study tumor growth and angiogenesis in mice who received subcutaneous injections of B16 melanoma cells in the ear.Their PAM showed irregular and linear vascular patterns, likely representing neovascularization of the dermis.The RCM enface image illustrated widespread pagetoid cells with cytologic atypia and nucleated cells within the dermal papilla.The PAM provided significant contrast and penetration depth, based on optical   absorption properties, and visualized vascularity and pigmentation, whereas RCM illustrated cytological features.On the other hand, Xu et al. 134 developed an integrated OR/AR-PAM system for multiscale imaging capability with high-speed wide-field imaging based on a polygon scanner.The polygon has six aluminum-coated surfaces that reflect the light and the acoustic beams: in this way six repeated cross-sectional scans can be obtained in one rotation of the motor, increasing image acquisition speed.With the two modalities and using two different wavelengths (532 and 1064 nm) a sub-CM is detected and separated from the surrounding microvasculature [see Fig. 6(e)].

Human studies
Recently, angiogenesis of human melanomas has been imaged by He et al. 109   10 dysplastic nevi and 10 melanomas, a quantification of the vasculature features was performed to identify biomarkers for melanoma detection.A total of six biomarkers were calculated: the total blood volume, vessel density, average vessel length, tortuosity, fractal number, and lacunarity.It was found that those markers showed differences between malignant and benign lesions, supporting the possibility to use this system to improve melanoma diagnosis.

Lymph Node Metastases
Melanoma survival rates are high when the disease is caught early, but decrease significantly after nodal or distant metastasis.The sentinel lymph node (SLN) is the hypothetical first lymph node draining the cancer; SLN metastasis has been shown to predict the pathologic state of the nodal basin. 11An SLN biopsy is typically performed during wide local excision if the Breslow depth is >1 mm; moreover, SLN biopsies are considered in ulcerated tumors of any depth. 11,255he SLN for a specific melanoma is identified through pre-operative lymphoscintigraphy, or intra-operatively using either a blue dye injection near the primary tumor site or a gamma probe with technetium-99 sulfur colloid. 11After excision, SLNs are examined via histopathology and immunohistochemistry for metastasis.However, these examinations can miss the presence of metastases due to false negative rates varying from 5% to 21%. 256Once a lymph node is removed, typically 6 to 10 sections of ∼6 μm thickness are taken and examined for metastasis.Thus, in a typical node with 1 cm length, there are hundreds of possible sections, only a fraction of which are histologically examined. 176On the other hand, the presence of metastasis in an excised node can be determined by PAI 257 since most melanomas are highly melanotic, containing ∼95% melanin, 11 and melanin is a highly absorbing chromophore in NIR range. 115Therefore, SLN metastases can be accurately identified by PAI, leading to appropriate selection of sections for histological examination, mitigating the possibility of false negative results. 258

Animal studies
PAI has been studied for the imaging of lymph nodes (Table 3) to identify metastases both ex vivo (postsurgical resection) and in vivo.McCormack et al. 176 used a three-single-element-transducer based PAT prior to and after injecting a human melanoma cell line (HS 936) into ex vivo lymph nodes from a healthy canine and pig.The PAT system they used consisted of a 600 μm diameter optical fiber connected to a tunable laser and an acoustic sensor made from polyvinylidene fluoride film.The 532 nm wavelength light was illuminated from the top of the lymph node, and the PA signals were detected from the bottom [see Fig. 7(a)].The control lymph nodes (with no injected melanoma cells) showed no PA response [see Fig. 7(a)(i)], whereas the melanoma cells in the excised lymph nodes generated PA signals as shown with the arrow in Fig. 7(a)(ii).A pig lymph node with only 500 injected melanoma cells and a 100 to 200 μm diameter lymph node produced a PA response.Neuschmelting et al. 180 utilized a multispectral (wavelengths 700 to 860 sampled) PAT system with a cylindrically focused transducer array (iThera Medical) and compared it with fluorodexyglucose (FDG) positron emission tomography (PET)/CT for imaging B16F10-derived melanoma micrometastases and macrometastases to lymph nodes and in-transit metastases (metastases moving from the primary tumor to the nearby nodal basin) in mice in vivo.The PAT system detected lymph node micrometastases in the cortical region of lymph nodes (after 2 weeks' tumor cell inoculation) that were too small for FDG PET/CT to detect.It also delineated in-transit metastases, which were observed as bright clusters between the primary melanoma site and the nodal basin.Both PAT and PET/CT could detect macrometastases [see Fig. 7(b)], but only the PAT system unmixed signals to enable detection of micrometastatic infiltration of melanoma in the cortex of popliteal nodes (white dashed circles).This multispectral PAT system distinguished melanoma lymph node metastases from other neoplastic and nonneoplastic lymphadenopathies (due to melanin's contrast) but FDG PET/CT could not (due to nonspecific FDG uptake and relatively low resolution).Most recently, Sinnamon et al. utilized a Vevo LAZR PAT system to visualize inguinal lymph node metastases in vivo at 4 and 8 weeks after inoculation with B16 melanoma cells in the flank region in BRaf-PTEN transgenic mice. 174,183In total, 49 lymph nodes were imaged in 25 mice, with metastatic cells present in 17 lymph nodes (35%), with histopathological confirmation.Thus the system was able to image in vivo melanin PA signals of a lymph node containing melanoma metastasis at 60 days after  the induction of the tumor [see Fig. 7(c)(i)].The negative control mouse without tumor induction is shown in Fig. 7(c)(ii).The PA melanin signal within the positive lymph nodes was significantly higher than in the negative control mouse, with no difference in PA signals between the adjacent soft tissue of positive and negative lymph nodes.The strongest predictor of melanoma metastasis was the ratio of lymph node to soft tissue PA melanin peak signal.

Human studies
Many different PAI modalities have been utilized for ex vivo studies of metastases in melanoma SLNs, including LA-PAT and arc-shaped PAT. 117,169,174PAT has also been used for in vivo studies. 117The first human study on melanoma lymph node metastasis was conducted by Grootendorst et al. 169 using a curvilinear array.Patients with proven metastatic disease undergoing inguinal or axillary lymphadenectomy were enrolled, and 1 or 2 lymph nodes were randomly selected for ex vivo multispectral analysis.The PA laser was illuminated from the top of the sample and the US detector array was rotated 360 deg around the sample.A total of six lymph nodes were imaged, and PAT revealed that three lymph nodes were metastatic and three were benign.All three malignant nodes (LN1, LN2, and LN3) displayed increased PA signals (from melanoma cells), whereas all three benign nodes had a substantially weaker signal (signal likely from hemoglobin and possibly other chromophores) when imaged at wavelengths from 720 to 800 nm [see Fig. 8(a)].The results were confirmed through histopathology.Langhout et al. 174 used a LA-PAT system and imaged 12 lymph nodes.Histopathology revealed that three nodes were metastatic and nine were benign.Again, melanoma-positive nodes [see Fig.  lymph nodes (absent melanin deposits) allowed for computation of the entire nodal volume.The largest human study on PAI of melanoma metastasis to lymph nodes was carried out by Stoffels et al. 117

Metastases to the Blood: Imaging CTCs
CTCs originate in the primary tumor and acquire genetic and structural alterations, leading to changes in cellular signaling and protein expression.This results in bloodstream intravasation and possibly invasion to other organs to develop secondary tumors. 259,2602][263] CTCs can be detected via flow cytometry, which has traditionally involved detecting fluorescent signals from cells labeled with multicolor probes ex vivo, which is known as conventional flow cytometry. 264,265However, this method requires invasive blood extraction from patients and is limited in its ability to detect infrequent CTCs. 265dditional CTC assays have been developed, 266 but have not been studied on a large scale, limiting their clinical translatability.Flow cytometry can be done in vivo and noninvasively using PA flow cytometry (PAFC).The concept of PAFC is similar to PAI: an excitation light is absorbed by chromophores in blood cells, causing local thermoelastic expansion and subsequent production of acoustic waves, which can be detected by an US transducer.PAFC configurations are an adaptation of OR-PAM, as high-pulse-repetition-rate lasers are also used in PAFC to generate detectable PA signals from individual CTCs. 265Higher pulse rates improve the signal-to-noise ratio (SNR) through averaging PA signals from individual CTCs; SNR is determined by the ratio of PA signals from individual CTCs to signals from other blood cells and background noise. 265The main chromophore in blood cells is hemoglobin in red blood cells (RBCs), but the main chromophore in melanoma CTCs is melanin.As with many other PAI modalities, in PAFC, multispectral analysis is often used to differentiate hemoglobin/melanin signals in the vasculature.Thus, unmixing of multispectral PA signals can allow for differentiation of CTC signals from oxy-and deoxy-hemoglobin. 167179,189,190

Animal studies
PAFC has demonstrated the ability to detect unlabeled (and labeled) CTCs in the mouse vasculature after injection of CTCs in the tail vein or from inoculation of primary melanoma tumors in the skin or ears. 87,167,168,172,189,190Overall, PAFC accurately detected CTCs from the background RBCs in the mouse vasculature, and PAFC signals increased as metastasis increased. 139,167,168,171,172,189,190Deán-Ben et al. 191 employed spherical array PAT for real-time visualization of passage and trapping of individual B16 melanoma cells in the whole mouse brain.Imaging was performed with the laser wavelength and pulse repetition rate set to 700 nm and 50 Hz, respectively.About 100 frames of PA images were acquired before injection of B16 melanoma cells and averaged to determine a baseline.The system could identify and track B16 melanoma cells after injection by taking the difference between before and after images. 191

Human studies
Galanzha et al. 166 studied CTCs in healthy patients and patients with melanoma using PAFC in vivo.PAFC data from healthy patients were used to calculate false positives and to study PA artifacts. 166PAFC accurately identified unlabeled CTCs in 15/16 melanoma patients. 166The system uses MPAI with one channel for detection of blood and a second channel for melanin.The channels are shown individually and fused together in Fig. 9(a)(i), and fused images showing movement of melanoma CTCs in an artery [Fig.9(a)(ii)] and a vein [Fig.9(a)(iii)] are also presented.This system has relatively poor lateral resolution (Table 4), which the authors explain is caused by light beam blurring. 166Recently, PAT systems have also been used to detect CTCs.Hai et al. 171,192 employed a LA-PAT system to detect melanoma CTCs in patients in vivo.Based on the optical absorption coefficient ratio, an excitation wavelength of 680 nm was chosen to maximize the contrast between melanoma CTCs and blood and achieve the highest detection sensitivity.Contrast-to-noise ratio (CNR) was used to quantify melanoma CTCs from the background tissue (RBCs).From the imaging session of the forearm of a positive patient with stage IV metastatic melanoma, a CTC was detected with a CNR of 9.4 [see Fig. 9(b)].The single cells were captured at five frames and the flow speed was estimated to be 9.6 mm∕s.They imagined 16 stage III and IV melanoma patients and successfully detected suspected melanoma CTCs in three patients.In fact, two of the three had disease progression, but four of those found CTC-negative also had disease progression.The lower rate of CTC detection by LA-PAT compared with PAFC could be attributed to the use of melanin as the single marker to identify and detect CTCs, and the fact that the patients were measured at only one clinical time point instead of on multiple repeat visits. 171,192Also, in the previous study, optical clearing techniques, including      microdermabrasion and glycerol sonophoresis, increased PA signals by two-to threefold, due to the reduced light scattering in superficial skin layers. 166PAT has tremendous clinical potential in imaging CTCs and disease monitoring in melanoma, given the fact that PAT can image very deep in the tissue with very high optical contrast.Moreover, utilization of contrast agents for molecular PAI can further increase the sensitivity of PAT to imaging cancer cells that do not express melanin.

Virtual Histology
Recently, all-optical PA microscopy in reflection-mode has been shown to be able to form histology-like images of various cancers on unstained slides to distinguish tissue types. 142,267The principle behind this technology is that by concurrently measuring radiative and nonradiative (in the form of acoustic) emissions from tissue samples illuminated with light in the UV range (266 nm), it is possible to differentiate DNA, RNA, collagen, and elastin, among other chromophores.Nuclear contrast, in particular, comes from relaxation of DNA, and nonnuclear contrast from relation of extranuclear proteins.These enable high-resolution images remarkably analogous to traditional chemical haematoxylin and eosin (H&E) staining. 267,268This technique has very recently been applied to the analysis of skin biopsies suspected for melanoma. 269,2706 Exogenous Contrast Agents for Melanoma Detection PAI most commonly utilizes intrinsic (endogenous) contrast agents, such as melanin and oxyand deoxy hemoglobin to assess melanomas.271 However, an exogenous, melanoma-specific contrast agent could assist in assessing early-stage melanomas, tumor staging, surgical excision planning, detection of lymph node metastases, and detection of CTCs.In Table 5, some of these applications are demonstrated.Gold nanoparticles (AuNPs), due to their intrinsic bioinertness and highly tunable optical properties, have shown remarkable promise in cancer diagnostics and treatment.273 As a contrast agent, AuNPs display optical absorption and scattering cross-sections that are greater than those of organic dyes, making them appropriate contrast agents for several applications in biomedical optics, including PAI.For example, gold nanorods (AuNRs) have been used to increase optical contrast in CTCs.272,274 Zharov et al. 272 used PAFC to detect CTCs (human squamous carcinoma cell line SQ20B) labeled with AuNRs, which were 15 nm × 52 nm in size and had a maximum absorption at 840 nm. Thecancer cells labeled with AuNRs were injected into the mouse circulatory system through the tail vein.The real-time accumulation of AuNRs in the cells was monitored through an increase in PA signal.The maximum absorption contrast introduced by AuNRs compared with background tissue was ∼29∶1.The capability of AuNR was compared with that of a conventional contrast agent called ICG.At the same laser energy, linear PA signals from AuNR-labeled cancer cells were five to seven times stronger than the signals from cancer cells stained with ICG.

Animal studies
Kim et al. 173 studied PA skin imaging of gold nanocrystals (AuNCs) bio-conjugated to ½Nle 4 ; D-Phe 7 -α-melanocyte-stimulating hormone (½Nle 4 ; D-Phe 7 -α-MSH) and compared them to poly(ethylene glycol) (PEG)-AuNCs.Melanoma cells strongly overexpress α-MSH receptors.In their study, mice were inoculated with B16 melanoma cells and either one or the other contrast agent was injected via tail vein.In vitro studies showed that melanoma cellular uptake of ½Nle 4 ; D-Phe 7 -α-MSH-AuNCs was ∼3.5 times greater than PEG-AuNCs uptake after 6-and 24-h incubation periods.In vivo experiments involved two groups of mice: one group (n ¼ 4) received ½Nle 4 ; D-Phe 7 -α-MSH-AuNCs and the other group (n ¼ 4) received PEG-AuNCs, both via tail vein [see Fig. 10(a)].Six hours after injection, the PA signals of ½Nle 4 ; D-Phe 7 -α-MSH AuNCs increased 38%, whereas the PEG-AuNCs increased only 13% [see Fig. 10(a)(iv)].The number of AuNCs was quantified in the excised tumors using inductively coupled plasma mass spectrometry.The mean number of ½Nle 4 ; D-Phe 7 -α-MSH AuNCs per gram of tumor was found to be 360% times greater than that of PEG-AuNCs, indicating the extremely high uptake of ½Nle 4 ; D-Phe 7 -α-MSH by melanoma cells.More recently, Li et al. 175 evaluated a nanoparticle composed of AuNRs and liquid perfluorocarbon (PFH) and conjugated it to a monoclonal antibody to melanoma-associated antigens (MAGE-1 antibody) and used an LA-PAT system for imaging.In vitro experiments showed a high number of MAGE-Au-PFH-NPs concentrated in the plasma membrane and cytoplasm of melanoma cells, compared with Au-PFH-NPs (control), which only showed a weak binding around tumor cells.Laser irradiation of nanoparticles with PFH results in a phase change (liquid PFH to gas microbubbles), increasing the acoustic impedance of surrounding tissues and subsequently causing a signal enhancement.In vivo PAI in tumor-bearing mice revealed a significantly higher PA signal intensity 2 h after MAGE-Au-PFH-NPs injection into the tail vein compared to Au-PFH-NPs; higher conjugate concentrations produced greater PA signals [see Fig. 10(b)].
Wu et al. 195 developed cell membrane-camouflaging hollow copper sulfide nanoparticles (ID-HCuSNP) by coating the membrane of melanoma cells with doxorubicin and ICG-loaded hollow copper sulfide NPs to enhance their targeting ability.They injected a mouse model with ID-HCuSNP and after 4 h, and they observed a strong local PA signal in the tumor area due to the accumulated NPs. 195This showed cancer cell membrane-camouflaged NPs having an excellent  self-recognition ability to the aimed tumor cells in vivo [see Fig. 10(c)].Galanzha et al. 165 used PAT with mapping techniques (multicolor PA lymph flow cytometry, PA lymphography, and absorption image cytometry) to examine metastasis to the prenodal lymph vessels and SLNs in mice ex vivo and in vivo.Specifically, after a visible primary melanoma tumor had developed in the mouse ear, they injected magnetic nanoparticles (MNPs) that provided stronger PA signals from lymphatics and SLNs at 639 nm.However, similar NIR absorption spectra of MNPs and melanin made it difficult for spectral identification of melanoma metastasis in the presence of MNPs.To resolve this problem, they used Evans Blue (EB) dye, which absorbs poorly at 639 nm only, while the presence of melanoma cells in lymphatics and in a node was identified through the appearance of PA signals at 850 nm.Because absorption of melanin also occurred at 639 nm above the background from EB dye, they used specific ratio of PA signals at 639 and 850 nm for additional identification of melanoma cells.They injected EB dye near melanomas derived from B16F10 cells and imaged the region in week 1 and 2 postinoculation.The number of metastatic melanoma cells in transit increased from 0.26 cells∕ min in week 1 postinoculation to 2.13 cells∕ min in week 2 postinoculation.The percentage of PA signal covering the examined SLN area increased from 6% in week 1 postinoculation to 39% in week 2 postinoculation.In the histology results, lymph nodes showed no metastases after 1-week inoculation and single metastases after 2 weeks inoculation.Thus the PA system was able to detect early micrometastases that could not be visualized in histology, illustrating its ability to detect even single metastatic cells (at week 1 postinoculation).
Despite the number of reports on the successful demonstration of Au nanomaterials for cancer theranostics, 275,276 accumulations in the liver and spleen due to resident macrophages that form the mononuclear phagocyte system are still an issue. 277,278Armanetti et al. 196   administration by exploiting the PA properties of AuNP-enriched ECFCs.They demonstrated in vitro that AuNP-loaded ECFCs are able to generate higher PA signals than AuNPs alone and also display spectral fingerprints that enable a reliable detection of labeled cells following intravenous injection. 196oxicity is always a concern when developing exogenous contrast agents.Factors for assessing biocompatibility include inertness, metabolism, and effective clearance rates.Material composition, surface modifications, shape, and size must all be designed carefully to maximize biocompatibility.The growing field of PAI for cancer diagnosis and treatment planning is accelerating research in biocompatible exogenous contrast agents. 279,280

Discussion
The scalability of PAI technology (from OR-PAM to AR-PAM to PAT) allows a wide range of applications including melanoma detection and depth determination, tumor-related angiogenesis, lymph node metastases, and presence of CTCs, all either with endogenous or exogenous contrast agents.Although these applications have been explored using small animal models for a couple dozen years, in the past decade or so, human observational studies have demonstrated the clinical potential for these systems.Although these technologies need more confirmational human studies, they lay a strong foundation for the near-term translation of PAI systems to the clinic for melanoma disease detection and management.
In terms of initial diagnosis and melanoma depth determination, if melanoma is in its very earliest stages (at the epidermis), OR-PAM provides rich information on tumor presence and angiogenesis-therefore, it can be used not only for detection but for accurate treatment planning, as the extent of angiogenesis may predict tumor aggressiveness.AR-PAM could be a better choice for deeper melanomas (approximately ≥1 mm).Although PAT could be implemented using ultrahigh frequency transducers, the use of it to detect and characterize early stage melanomas has not been explored, perhaps due to the requirement for a much-more sophisticated data acquisition system, transducer focal length, and complex light illumination configuration.Another shortcoming of PAT is that it is limited to providing 2D images, unless it is implemented with a scanning system, which adds complexity.Nevertheless, tunable lasers with a wide range of wavelengths (to accurately distinguish melanoma biomarkers such as melanin) are much more accessible for PAT implementation compared to AR/OR-PAM.Therefore, where permitted, high or ultrahigh frequency PAT could be a good choice for melanoma detection and depth determination.Complementing current histopathology of excised tissue, recently, all-optical PA microscopy in reflection-mode has been shown to form histology-like images of various cancers on unstained slides to distinguish tissue types.This technique has great potential to be in the clinic for rapid dermatological tissue analysis.Once a melanoma is appropriately detected, knowing whether it has metastasized to the SLN is the next important question for treatment planning.Because of the depth of SLNs, PAT might be the best choice, and in fact, in Table 3, all of the human studies of SLNs were performed by PAT, although in some cases, AR-PAM could potentially be used as well.In cases where melanoma is confirmed metastasized to the SLN (or beyond), detection of CTCs is very valuable for monitoring responses to cancer treatment and evaluating prognosis.With PAI being a technology based on high-energy laser, fast switching between imaging and ablation of CTCs has been investigated in small animals, it could be translated into a theranostic application, once the concept is sufficiently confirmed through clinical studies.
Although melanin is an adequate optical contrast for early-stage melanomas, it is not sufficient for later stage melanomas due to the limitation of light penetration in deep structures.Exogenous contrast agents have been shown to increase PA signals beyond the signal level obtained using the label-free PAI (endogenous contrast agents).As discussed in Sec.3.5, different tumor cell targets (antigens) of the antibody-coupled exogenous contrast agents have been tested on animal models.While the results seemed promising, exogenous contrast agents need to be proven safe to have clinical utility.In addition, before exogenous contrast agents are studied in humans, a unique target protein present on melanoma cells and absent on healthy cells will need to be identified, such as galectin-3 or collagen XVII, which have recently been shown to be overexpressed in melanoma cells. 281Furthermore, few if any studies have investigated whether a contrast agent could be applied topically on the melanoma lesion as opposed to intravenously, which could mitigate potential toxicity.However, PAI will likely need to gain clinical utility before exogenous contrast agents are rigorously investigated in humans.
While PAI holds great clinical potential, safety and cost are factors that may limit the ease of clinical translatability.There is a safety concern about tissue damage from the light excitation.The American National Standards Institute has already defined the maximum permissible exposure, which is the level of electromagnetic radiation that a person can be exposed to without harmful effects, for skin imaging; 91 another is potential damage to the eyes of the patient or the physician from stray laser light, which can be mitigated by using appropriate protective eyewear.Cost is a further potential limitation that can be mitigated by mass production of PAI systems.A final hurdle is the need for pulsed nanosecond lasers, which can be costly.][284] In recent years, there has been a growing interest among various companies in the development and commercialization of PAI systems for medical applications. 145,285Among them are iThera Medical GmbH, 105,130,180,[286][287][288][289][290][291][292][293][294][295][296][297] FUJIFILM VisualSonics, 135,171,187,298 ENDRA Life Sciences Inc., [299][300][301][302][303] TomoWave Laboratories, [304][305][306][307] and Seno Medical Instruments. 308,309lthough there exist commercial systems for skin imaging, there has not been any customized clinical system for melanoma imaging applications.

Conclusions
Prior CM studies in animals and humans have illustrated PAI's potential use in identifying tumor depth, analysis of angiogenesis, detection of lymph node metastasis, detection and ablation of CTCs, and ability to generate histology-like images of various cancers on unstained slides.These applications stem from the high spatial and temporal resolution, adequate penetration depth, and label-free nature of this imaging modality.
Many of these opportunities are based on the use of melanin as a high-absorbing chromophore.However, reliance on melanin has a few limitations.First, as noted above, 251 for tumors with very high density of melanoma, it can be difficult for PAI to penetrate through the tumor to detect lower boundaries without assistance from, for example, US structural information.Second, amelanotic melanoma, which accounts for 1% to 8% of all melanomas, is a form of melanoma with little to no pigment (lesion pigment not differentiated from skin). 310Methods focused on melanin detection could miss these lesions, and PAFC likely will miss amelanotic CTCs.
Despite the advantages of PAI described in this review, this technique has not yet been used as a diagnostic-assistant tool in large-scale clinical studies.One possible way to enroll large groups of patients in future studies would be to incorporate PAI before performing procedures to remove lesions suspicious for melanoma or before lymph node excisions.This would allow for characterization of PAI images from various pigmented lesions that may clinically resemble melanoma, such as benign nevi, dysplastic nevi, lentigo, etc., as well potential malignant lymph nodes.As more images are collected and analyzed, algorithms on the recognition of melanoma in PA images will mature.
Safety and cost are two key parameters in clinical translatability of PAI.The use of laser light must be limited to the maximum permissible levels for skin imaging, and protective eyewear or confining apparatus can mitigate potential damage to eyes.PAI system costs can be reduced by using a combination of LEDs and development of low-cost US transducers designed specifically for PA applications.

Fig. 3
Fig. 3 Hardware designs of selected PAI systems.(a) For melanoma detection and depth measurement: (i) all optical OR-PAM combined with OCT system.SLD, super luminescent diode; PD, photodetector; FC, fiber coupler; G, grating; GM, scanning galvanometer; C 1 , C 2 , and C 3 , collimators; L 1 and L 2 : lenses; M, mirror; DM, dichroic mirror; FPGA, field programmable gate array; ADC, analog-to-digital converter; OL, objective lens; S, sample; OCT, optical coherence tomography; AOPA, all-optical integrated photoacoustic.Reproduced from Ref. 132.(ii) Multiwavelength AR-PAM system.Reproduced from Ref. 113.(iii) 3D wide-field multispectral photoacoustic imaging of human melanomas, a dual-modal photoacoustic and US imaging system.Reproduced from Ref. 133.(iv) Multiscale PAM.NDF, neutral density filter; DM, dichroic mirror; M 1 and M 2 , mirrors; AD, achromatic doublet; P, prism; AL, acoustic lens; UT, US transducer; PS, polygon-scanning mirror, DAQ, data acquisition.Reproduced from Ref. 134.(v) Handheld photoacoustic system for melanoma imaging.Reproduced from Ref. 135.(b) Melanoma detection and angiogenesis: (i) multiscale PAM.NDF, neutral density filter; DM, dichroic mirror; M 1 and M 2 , mirrors; AD, achromatic doublet; P, prism; AL, acoustic lens; UT, US transducer; PS, polygon-scanning mirror, DAQ, data acquisition.Reproduced from Ref. 134.(ii) Schematic of faster raster scanning optoacoustic mesoscopy (RSOM).Reproduced from Ref. 109.(iii) Multiwavelength OR-PAM system.OL, objective lens; NDF, neutral dispersion fiber; MLPF, motorized long-pass filter; built a hybrid PA/US system with a sound-light coaxial/confocal design by punching a 2 mm diameter hole in the center of the transducer to deliver the laser light.Melanomas in mice were imaged in vivo at day 7 [see Fig.4(d)Ii)] and day 30 [see Fig. 4(d)(ii)] after B16 cell injection.A clear growth in tumor size and depth is observed as shown in B-scan images of melanoma at day 7 [see Fig. 4(d)(iii)] and day 30 [see Fig. 4(d)(iv)].Moreover, PAI visualized microvasculature around the tumor.Another experiment with mice was conducted by Wang et al. 251 using a dual-wavelength AR-PAM with visible and NIR light combined with US to image sub-CM.The fused images from the two wavelengths enrich the imaging information and allow more accurate detection of the melanoma, differentiating it from normal tissue.With the 3D distribution the boundary detection of the melanoma is easier and accurate and could be further enhanced by the US structural information helping the identification of the tissue boundaries and precisely locating the sub-CM [see Figs.4(e)(i)-4(e)(iii)].

5
5(b)(i)].The amplitude of the PA signal corresponds to the amount of melanocytes in the local area.The PA signals in melanoma, indicated by the white triangles [see Fig. 5(b)(ii)], were predominantly generated by optical wavelengths of 800 and 1064 nm.In contrast, the PA signals from the marking pen regions indicated by the white arrows were predominantly generated from

Fig. 4
Fig. 4 Melanoma detection and depth measurement in live animal models using different PAI configurations.(a) PA images of melanoma and vascular distribution in nude mouse skin.(i), (ii) En face PA images for the NIR light source (λ ¼ 764 nm) and visible light source (λ ¼ 584 nm) respectively: 1, melanoma; 2, vessels perpendicular to imaging plane; 3, vessels horizontal to imaging plane; 4, skin.(iii), (iv) B-Scan PA images along the red line in panels (i) and (ii).Reproduced from Ref. 116.(b) PA image of the melanoma showing both the top and bottom boundaries in nude mice in vivo.The red dots outline the melanoma.Reproduced from Ref. 188.(c) LA-PAT images of melanoma acquired in nude mice on day 3 (i) and day 6 (ii) after tumor implantation.Reproduced from Ref. 187.(d) PA maximum amplitude projection (MAP) images of melanoma in mice at (i) day 7, (ii) day 30 and PA B-scan images on (iii) day 7 and (iv) day 30.Reproduced from Ref. 184.(e) Fused 3D visible light and NIR PA MAP images in mice with melanoma.(i) visible light and US, (ii) NIR light and US, and (iii) visible and NIR light.Reproduced from Ref. 251.

Fig. 5
Fig. 5 Melanoma detection and depth measurement in human studies using different PAI configurations.(a) PAT of melanoma of two patients: (i) melanoma image acquired with a PA depth of 1.9 mm (cPA depth ¼ 1.67 mm); (ii) cutaneous melanoma metastasis in a patient lower leg; (iii) histology of the excised melanoma, showing actual Breslow depth of 1.67 mm; (iv) PAT melanoma image of the acral lentiginous melanoma with PA depth of 0.70 mm (cPA depth ¼ 0.62 mm); (v) acral lentiginous melanoma (data not shown: pBD ¼ 0.48 mm); and (vi) histology after complete excision, with actual BD of 0.78 mm.BD, Breslow depth; cPA, corrected photoacoustic depth; PAT, photoacoustic tomography; pBD, provisional Breslow depth.Reproduced from Ref. 135.(b) (i) Photograph and (ii) ex vivo PA image of excised melanoma tissue from a male patient.The melanoma regions are represented by dark red to bright-yellow color (white triangles), and the marking-pen regions are represented by dark green to bright-green color (white arrows).Yellow arrows indicate possible melanomas not found by histology.Reproduced with permission from Ref. 177.(c) PA image of in situ melanoma on upper left extremity on a patient using LA-PAT.Reproduced with permission from Ref. 115.(d) Measurement of PA depth of a nodular type of melanoma.(i) Photoacoustic MAP; (ii) photoacoustic MAP, overlaid with US image; and (iii)-(iv) photoacoustic unmixed and photoacoustic unmixed overlaid with US images, respectively.Blue arrows indicate the melanoma invasion and yellow arrows are the bottom boundary of the melanoma.MAP, maximum amplitude projection.Reproduced from Ref. 133.(e) Representative PA signals from human subjects with either (i), (iii) pigmented and (ii), (iv) vascular lesions at 422 and 530 nm, respectively.Reproduced with permission from Ref. 252.

Fig. 6
Fig. 6 Angiogenesis studies in melanoma identification.(a) PA imaging of melanoma tumor vasculature in mice using (i) 50 MHz and (ii) 100 MHz detector.Reproduced with permission from Ref. 182.(b) PA images showing tumor growth over time.PA images of the tumor area acquired at (i) day 2 (D02), (ii) day 4 (D04), (iii) day 7 (D07), and (iv) day 9 (D09) after injection of melanoma cells, the thick arrow points to the tumor.The inset in every image shows the same region, from an area very close to the tumor, and shows two large vessels (the inset arrow points to that region), between which small vessels grow over time.Reproduced with permission from Ref. 182.(c) PA vascular imaging of melanoma in mouse at (i) day 9, (ii) day 13, (iii) day 14, and (iv) day 15.Reproduced with permission from Ref. 185.(d) PA images of human melanoma in vivo for three scanned regions: scan 1: inside the lesion, scan 2: boundary of the lesion, and scan 3: outside the lesion.(i)-(iii) Maximum amplitude projection (MAP) cross-sectional faster raster-scan optoacoustic microscopic (FRSOM) images.(iv)-(vi) MAP images in the coronal direction corresponding to the epidermal layer in panels (i)-(iii).(vii)-(ix) Coronal images corresponding to the dermal layer.EP, epidermal; DR, dermal.Reproduced with permission from Ref. 109.(e) In vivo OR and AR images of melanoma in mouse using multiscale PAM.(i) Top and side view MAP images obtained by OR-PAM at 532 nm.(ii) Top and side view MAP images obtained by AR-PAM at 1064 nm.(iii) Merged images of top and side view from OR and AR-PAM.Reproduced with permission from Ref. 134.OR, optical resolution; AR, acoustic resolution.
8(b)(i)] displayed different PA signals (depth image) than benign nodes [see Fig. 8(b)(ii)] due to the difference in melanin distribution.Additionally, total volume imaging to the depth of 2 cm in benign

Fig. 7
Fig. 7 Animal studies of lymph node metastases analyzed by PAI.(a) Photoacoustic response from lymph nodes (i) without melanoma, the detector shows no signal with a noise floor of 100 μV, and (ii) with melanoma, showing a photoacoustic wave at 5 μs.Reproduced with permission from Ref. 176.(b) Comparative results between multispectral PAT and FDG-PET/CT systems for detection of macrometastasis in right popliteal lymph node in mice.The red circle encloses the melanoma macrometastasis region.White circle enclosed the contralateral popliteal healthy control node.Multispectral images were unmixed for deoxyhemoglobin (blue), oxyhemoglobin (red) and melanin (yellow).Fb, femur; Tv, tail vessel; Rc, rectum; Ut, urethra.Reproduced with permission from Ref. 180.(c) In vivo images of melanin-specific photoacoustic signal with grayscale underlay of a lymph node that (i) shows metastasis at 60 days post tumor induction and (ii) negative control mouse that did not undergo tumor induction.MPAI, multispectral photoacoustic imaging; FDG-PET/CT, fluorodexyglucose PET/CT.Reproduced with permission from Ref. 183.

Fig. 8
Fig. 8 Ex vivo and in vivo studies of human lymph node metastases analyzed by PAI.(a) PA signal strength of the selected areas within the lymph nodes (LN) at different illumination wavelengths.Reproduced with permission from Ref. 169.(b) Images of two human nodes.(i) PA image of a metastatic node, (ii) PA image of a benign node.Absence of PA signal deeper in the malignant node [as indicated by * in panel (b)] seems to be caused by the strong absorption by the melanin in the superficial area of the node.Reproduced with permission from Ref. 174.(c) (i) Lateral MIP and (ii) 3D rendering image of ex vivo optoacoustic image of a human lymph node from a melanoma patient.Grayscale represents hemoglobin background and color bar overlay shows the multispectral resolved signals for melanin.Reproduced with permission from Ref. 117.

Fakhoury
Imaging of the carotid artery of mice 168 Develop in vivo lymph tests using the principles of flow cytometry.Calculated the CTC flow rate In vivo: Mice tail vein Detection of CTCs.About 82% of unlabeled CTC were detected CFmelan-oma cells tagged with AuNP 178 The AuNP tagged melanoma cells showed 34% greater PA signal than the untagged cells Mice tail vein, ears vessels, and abdominal vessels 190 Detection of CTCs in melanoma-bearing mice.PA signals were detected immediately after injection.Calculation of the CTC flow rate In vitro: Detection of CTCs spiked in blood samples.In vivo: Assessment of effect of optical clearing on mice and humans 179 Detection of melanoma cells in blood flow 40 × 1100 μm In vivo: Mice, subcutaneous injection, detection of CTCs in ear 189 Identification and count of RBCs and CTCs in the blood vessels In vivo: Mice, injection of B16 cells in jugular vein or carotid artery 172 Detection of CTCs and clusters traveling in blood vessels λ2: et al.: Photoacoustic imaging for cutaneous melanoma assessment. . .

1 PAFC
In vivo: Healthy humans and humans with melanoma 166 Detection of melanin-bearing CTCs in patients with melanoma.CTC flow rate was calculated.PAFC distinguished CTCs from circulating blood Humans with melanoma 170 Detection of CTCs.Signal from a single melanoma cell is detectable λ2: In vivo: CTCs in mice, rat tail artery 208 PA detection of induced metastatic melanoma λ2: 622 nm PAFC λ: 820 nm CF: 10 MHz NL In vivo: Mice artery after tail injection 211 Detection of strongly absorbing cells with positive contrast and negative contrast for fluorescence NE:

5 NL
In vivo and ex vivo: Mice brain212,215 Detection and counting of CTCs in CSF of tumor-bearing mice λ2: in vivo: Melanoma cells in mouse blood and in mice paw 138 Reliable detection of melanoma cells in the vessels after upstream arterial Human blood samples with stage I, II, and III melanomas 199 Quantification of CTCs in serial blood samples at early stage melanoma to predict metastatic disease In vitro: Bovine blood samples with melanoma cells 140 Detection of melanoma cells flowing in blood sample.Estimation of the flow speed of the cell in the fluid In vivo: Mice carotid artery 139 Visualization of CTCs in mice cortical arteries and veins and calculation of the CTC flow rate In vivo: Mice brain with melanoma CTCs 191 Detection and counting of individual CTCs in the CSF BW: 100% NE: 512 Note: RBC, red blood cell; λ, wavelength; CF, central frequency; BF, broadband frequency; BW, bandwidth; NE, number of elements; NL, not listed; AuNR, gold nanorods; LR, lateral resolution; AR, axial resolution; AR-PAFC, acoustic resolution PA flow cytometry; OR-PAFC, optical resolution PA flow cytometry; SIP-PAT, single pulse panoramic PAT; PVDF, polyvinyledene fluoride; CNR, contrast-to-noise ratio; CSF, cerebral spinal fluid.

Fig. 9
Fig. 9 Imaging melanoma circulating tumor cells in vivo.(a) PA flow cytometry: channels shown individually and fused together (i), fused images showing movement of melanoma CTCs in an artery over time (ii), and in a vein (iii).(b) LA-PAT system uses differential analysis of images taken over time to find moving CTC cells: (i) PA snapshots of the melanoma CTC in the patient.The yellow arrows indicate structures, including the skin, vessel boundaries, and subcutaneous fat layer.The red arrows highlight the melanoma CTC.(ii) Differential PA images showing only the melanoma CTC.(iii) Differential PA images superimposed on structural images, highlighting the melanoma CTCs.Reproduced with permission from Ref. 192.
utilized endothelial colony forming cells (ECFCs) to carry AuNPs and explored the antitumor effects and the tumor-homing efficiency following single intravenous injection into tumor-bearing mice.They assessed AuNPs biodistribution in freshly excised mice organs at different time points post

Fig. 10
Fig. 10 In vivo animal studies involving exogenous PAI contrast agents for melanoma detection.(a) In vivo MAP images of B16 melanomas using ½Nle4; D-Phe7-α-MSH-and PEG-AuNCs.(i), (ii) Photographs of tumor in mice before injection.(iii), (v), and (vii) Time course PA images after injection of ½Nle4; D-Phe7-α-MSH-AuNCs.(iv) (vi), and (viii) Time course PA images after injection of PEG-AuNCs.Reproduced with permission from Ref. 173.(b) PA images of B16 melanomas in mice at different time points after the injection of (i) MAGE-Au-PFH-NPs and (ii) AU-PFH-NPs.Reproduced with permission from Ref. 175.(c) PA imaging of melanoma in mice (i) before and (ii) after the injection of ID-HCuSNP@B16F10 NPs.Reproduced from Ref. 195.MAP, maximum amplitude projection.

Table 1
Specific characteristics of PAI systems used in published studies on melanoma detection/depth measurement.

Table 2
Summary of PAI studies on melanoma tumor angiogenesis.

Table 3
Summary of ex vivo and in vivo human and animal PAI studies for detection of melanoma metastasis to lymph nodes.
The SLN was detected in the rat beneath 2.2 cm thick chicken tissue layer, at a depth that is at least typical of SLN in humans BW: 90% NE: 192 Note: λ, wavelength; F, fluence; CF, central frequency; BW, bandwidth; NE, number of elements; NL, not listed; LA-PAT, linear array photoacoustic tomography; PAFC, photoacoustic flow cytometry; PAF, photoacoustic finder; AR-PAM, acoustic resolution photoacoustic microscopy; OR-PAM, optical resolution photoacoustic microscopy; SLN, sentinel lymph node.
using a multispectral arc-shaped PAT.They analyzed 506 SLNs from 214 patients with a Breslow depth of at least 1 mm: 148 SLNs (from 65 patients) were analyzed by multispectral PAT ex vivo and histology, whereas the other 358 SLNs (from 149 patients) were analyzed by the conventional European Organization for Research and Treatment of Cancer (EORTC) melanoma group protocol.Their system detected metastases in 22.9% of excised SLNs compared with 14.2% by the EORTC Melanoma Group protocol.Ex vivo analysis by PAT showed 100% sensitivity and 62% specificity.Then, an in vivo experiment was carried out using PAT and indocyanine green (ICG) (a NIR fluorophore injected peri-tumorally) as a contrast agent to image 41 SLNs in 20 patients.PAT visualized ICG-marked SLNs to the depth of 5 cm (discerned by single photon emission computed tomography/CT) and with 100% concordance with the gold standard of SLN detection, 99 m Tc-nanocolloid-guided lymphoscintigraphy.In vivo PAT analysis revealed a sensitivity of 100% and specificity of 48.6%.With 100% sensitivity, both ex vivo and in vivo PAT identified noncancerous SLNs in 189 total lymph nodes without any false negatives but with a high rate of false positives.The quantification of melanin was performed with multispectral analysis and then correlated with the localization of metastatic cells [see Fig.8(c)].

Table 4
Summary of ex vivo and in vivo human and animal PAI studies on melanoma CTCs.

Table 5
Use of exogenous PA contrast agents for melanoma detection in mice.